The endocannabinoid anandamide (N-arachidonoylethanolamine, AEA) activates type-1 (CB1R) and type-2 (CB2R) cannabinoid receptors [Howlett et al., 2004]. CB1R is localized mainly in the central nervous system, but is also expressed in peripheral tissues like immune cells. Conversely CB2R is predominantly expressed peripherally, but is also present in the brain. Therefore, activation of CB1 or CB2 receptors by AEA has many central and peripheral effects, that are terminated by cellular uptake through the AEA membrane transporter (AMT), followed by degradation to ethanolamine and arachidonic acid by the fatty acid amide hydrolase (FAAH) [Bari et al., 2006]. On the other hand, the checkpoint in AEA synthesis is the N-acyl-phosphatidylethanolamines (NAPE)-hydrolyzing phospholipase D (NAPE-PLD), which releases on demand AEA from membrane NAPEs [Fezza et al., 2005]. Together with AEA and other congeners like 2-arachidonoylglycerol, N-arachidonoyldopamine, noladin ether and virodhamine, the proteins that bind, transport, synthesize and hydrolyze these lipids form the “endocannabinoid system” [Bari et al., 2006]. Also the ability of AEA to bind to and activate type-1 vanilloid receptors (now called transient receptor potential channel vanilloid receptor subunit 1, TRPV1) has attracted growing interest, and in fact AEA is considered a true “endovanilloid”. Interestingly, activation of CB1R or of TRPV1 by AEA can exert opposite biological effects, for instance by protecting or inducing, respectively, apoptosis in neuronal and peripheral cells [Bari et al., 2006].
A still unresolved, though critical, issue in endocannabinoid research is the mechanism by which AEA enters the plasma membrane and is transported inside the cells [recently reviewed by Battista et al., 2005]. The existence of a selective AEA membrane transporter (AMT) has been postulated. However, the molecular identity of AMT remains unknown, and, at present, molecular probes to test its expression at the protein or messenger RNA level are not yet available. In addition, the kinetic features of AEA uptake do not rule out other mechanisms of transmembrane transport, being compatible, for example, with a simple diffusion process driven by FAAH-catalyzed hydrolysis of AEA. However pharmacological, biochemical and confocal microscopy studies strongly suggest that an authentic AEA membrane transporter exists, and that this is distinct from FAAH [Oddi et al., 2005].
The lack of cloning and expression of the transporter protein has prevented the development of molecular tools like oligonucleotides or antibodies, able to give definitive proof of the presence on the cell surface of an active membrane transporter for AEA (AMT) for AA or other equivalent fatty acids.
A recent report has identified a high-affinity binding site involved in the transport of endocannabinoids, by means of a potent, radiolabelled competitive inhibitor of AEA uptake: LY2318912 [Moore et al., 2005].
However, to date only one non-radioactive AEA analogue able to visualize AEA movement inside the cells by mean of fluorescence microscopy has been described [Muthian et al., 2000]. This compound, named SKM 4-45-1, is nonfluorescent until it is transported into the cells, where cytosolic esterases can activate its fluorescein moiety.
The mechanisms responsible for the AEA uptake and transport are not yet well characterized, primarily because of the lack of suitable molecular tools. In fact, although the current radiometric-based techniques using radiolabeled compounds such as [3H]AEA, [14C]AEA or [125I]LY2318912 yield a great sensitivity for the analysis of the kinetic features of AEA transport, they do not allow to visualize its specific pathways for internalization and intracellular targeting. Moreover, radioisotopes and radioimmunoassays used for these purposes, are not only very expensive, but arise problems due to the hazard of working with, and disposing of, radioactive materials.
On the other hand, SKM 4-45-1 is nonfluorescent in the extracellular environment and becomes fluorescent only when exposed to intracellular esterases. Therefore, its use is restricted to intracellular compartments of cells that express a sufficient esterase activity [Muthian et al., 2000], and is not suitable to visualize AMT on the cell surface [Oddi et al., 2005]. Another limitation of SKM 4-45-1 is that the manifestation of intracellular fluorescence occurs as a result of two kinetic processes, uptake and intracellular de-esterification. Therefore, the utility of SKM 4-45-1 in kinetic studies of AEA transport is extremely limited.
Furthermore, due to low signal intensity associated with direct fluorescence, SKM 4-45-1 does not prove of particular efficacy for the fine morphological analyses of the intracellular trafficking and metabolism of AEA [Muthian et al., 2000].
In order to shed some light on the metabolic regulation of AEA and related fatty acid amides activity, it would be very useful to avail means capable of visualizing through microscopy techniques the transport and intracellular trafficking of these lipids within cells and tissues.
Hence, a first scope of the present invention was to provide means suitable to trace AEA transport and trafficking by biochemical and morphological techniques.
Clinical/Diagnostic Relevance of Cannabinoid System
The impressive expansion of cannabinoid research in the past decade provided a large body of data regarding the potential involvement of endocannabinoid system in an ever-increasing number of pathological conditions, including neurological, cardiovascular, gastrointestinal, reproductive disorders, and cancer [Bari et al., 2006]. Therefore, alteration of the activity of one component of this system, such as the AEA membrane transporter, could have therapeutic value for the treatment of several human diseases.
Among endocannabinoids, anandamide (AEA) signaling has been involved in a number of physio-pathological conditions and has been attracted growing interest in pharmacology for its multiple diagnostic and/or therapeutic use, for example, in several disorders of central nervous system (CNS), in inflammatory conditions, pregnancy failure, pain treatment, and anxiety management [Bari et al., 2006]. Physiological experiments show, in fact, that AEA may be as important in regulating our brain functions in health and disease as other better-understood neurotransmitters, such as dopamine and serotonin. In particular, endogenous levels of AEA are elevated in the cerebrospinal fluid of Parkinson's disease and schizophrenic patients [Pisani et al., 2005]. In the latter subjects, AEA was supposed to be released in response to psychotic symptoms, in order to help their control, rather than to trigger psychosis. Furthermore, in a murine model of multiple sclerosis, the AEA content is altered in brain region involved in the disease and inhibition of endocannabinoid uptake significantly ameliorated spasticity. In this line, a recent article clearly demonstrates that cannabinoid type 1 receptors (CB1R) play an important role in neuroprotection by endogenous cannabinoids. Administration of kainic acid (KA), an excitoxin that induces neuronal seizures in vivo, rapidly increased hippocampal levels of AEA that induced protection against excitotoxicity [Bari et al., 2006].
In addition, several reports have shown that the activation of the central endocannabinoid system increases food intake and promotes weight gain. In genetic animal models of obesity, brain endocannabinoid levels are increased and CB1R is downregulated. Recently, an increase of circulating endocannabinoids levels has been reported in obese women. In this line Rimonabant (SR141716), a selective antagonist of CB1R developed by Sanofi-Aventis, has also been developed as an anti-obesity drug [Bari et al., 2006].
In peripheral systems, the detection of high levels of AEA in the uterus has been reported to indicate a high chance of miscarriage, which seems of great diagnostic interest and has screening potential. The important role of screening and quantifying AEA as a risk factor for cardiovascular disease and vascular dysfunction was also emphasized by other reports [Bari et al., 2006].
Therefore a simple, sensitive and cheap method to measure the concentration of AEA in biological fluids and tissues would be very useful and of diagnostic value. Its development could be possible by using b-AEA (MM22).
With respect to the potential therapeutic value of AEA, it has been found that administration of AEA provides relief in several models of neuropathic and inflammatory pain. Unfortunately, this compound is rapidly inactivated by enzymatic hydrolysis, which prevents its effective medical use. On the other hand, more metabolically stable agonists of cannabinoid receptors present unwanted psychotropic side effects, including memory impairment and catalepsy. It seems of interest that, as demonstrated in this invention, b-AEA is not hydrolyzed by FAAH and does not bind to CB or TRPV1 receptors.
Therefore, a further purpose of the invention was to provide a means to prolong the physiological effects of AEA, by controlling its uptake and the hydrolysis.